This invention relates to methods for cell manipulation and more specifically to methods for transiently permeabilizing a cell so that a variety of exogenous materials, such as expressible foreign DNA, can be loaded into the cell.
Previous loading methods have included chemical treatments, microinjection, electroporation and particle bombardment. However, these techniques can be time-consuming and suffer from low yields or poor cell survival. Another technique termed xe2x80x9coptoporationxe2x80x9d has used light directed toward cells and the surrounding media to induce shock waves, thereby causing small holes to form temporarily in the surface of nearby cells, allowing materials to non-specifically enter cells in the area. Another technique termed xe2x80x9coptoinjectionxe2x80x9d also uses light, but directs the light to specific cells. Nevertheless, previous light-based implementations techniques have suffered from the same disadvantages as other loading techniques.
Thus, there is a need for a method for rapid and efficient loading of a variety of exogenous molecules into cells, with high cell survival rates. The present invention satisfies this need and provides related advantages as well.
The present invention provides optoinjection methods for transiently permeabilizing a target cell. In the general method, the steps are (a) illuminating a population of cells contained in a frame; (b) detecting at least one property of light directed from the frame; (c) locating a target cell by the property of light; and (d) irradiating the target cell with a pulse of radiation.
In particular embodiments, a static representation is obtained when the population of cells is substantially stationary; the cells are illuminated through a lens having a numerical aperture of at most 0.5; the pulse of radiation has a diameter of at least 10 microns at the point of contact with the target cell; or the resulting pulse of radiation delivers at most 1 xcexcJ/xcexcm2. As a result, the method provides rapid and efficient loading of a variety of exogenous molecules into cells, with high cell survival rates.